The invention relates generally to the fields of microbiology and veterinary medicine. More particularly, the invention concerns compositions and methods relating to detecting Sarcocystis neurona. 
Equine Protozoal Myeloencephalitis (EPM) is a common cause of neurologic disease in New World horses. It is caused by a parasite termed Sarcocystis neurona (S. neurona), an obligatory intracellular apicomplexan parasite whose multi-phase life cycle is completed in either one or two hosts. S. neurona is known to cycle naturally between opossums and both none-banded armadillos and striped skunks. Horses typically become infected by consuming infectious parasite stages found in opossum feces. Once a horse has been infected, S. neurona can travel to the brain and spinal cord, where merozoite stages of this parasite replicate and cause pathology.
Horses with EPM typically present with lameness, but may alternatively or additionally present with symptoms characteristic of primary brain disease. Because the parasite can inhabit any area of the central nervous system (CNS) of the horse, symptoms associated with EPM can vary widely. The degree of infection can range from subtle to severe and can involve the brain and/or the spinal cord. EPM is usually progressive.
Presently, a definitive diagnosis of EPM is made by post-mortem examination, where S. neurona organisms are identified in histological lesions. The organ may also be cultured from the lesion. The presence of the organism in the histologic section or when cultured from the lesion establishes the diagnosis. Heretofore, pre-mortem methods for diagnosing EPM were based on assays using whole merozoites, and not a purified protein, to probe for the presence of anti-S. neurona antibodies (as an indication of infection) in the horses. The use of such whole merozoites results in significant cross-reaction with non-S. neurona specific antibodies (e.g., those against other Sarcocystis species). This cross-reactivity obscures interpretation of results using whole merozoite-based assays.
The invention relates to the discovery and characterization of a 29 kilodalton (kDa) protein found on the surface of merozoite stage S. neurona. This antigen, termed SnSAG-1 or SnSMA1, is an immunodominant antigen recognized on protein blots. Using purified or recombinant SnSAG-1 (i.e., rSnSAG-1) antigen, accurate assays for diagnosing EPM in horse pre-mortem have been developed. These assays involve identifying a marker indicative of the presence of the 29 kDa antigen or an antibody to this antigen in a sample to be tested. Thus, because a single purified antigen or marker is utilized in such assays, the cross-reactivity problems associated with whole-merozite based assays are obviated or much reduced.
A cDNA copy of the mRNA which encodes the SnSAG-1 antigen has been cloned from a gene library prepared from an isolate of S. neurona. The original clone was identified in a collection of random sequence tags prepared to characterize the cDNA library. Additional clones of the same gene sequence were obtained to identify a full length gene. The nucleotide sequence of a full-length gene clone was determined. This sequence or the clone itself can be used to gusto prepare the SnSAG-1 antigen in a recombinant or other synthetic form for use in diagnostic tests and vaccine development.
Accordingly, the invention features a composition for detecting the presence of S. neurona in a biological sample. In one variation, the composition includes a SnSAG-1 marker that is a purified nucleic acid including a nucleotide sequence that encodes a protein that shares at least 50% or at least 90% sequence identity with SEQ ID NO:1. In this variation, the nucleotide sequence can also encode the protein of SEQ ID NO:1. For example, the nucleotide sequence can be SEQ ID NO:3.
In a second variation of the composition, the SnSAG-1 marker is a purified polynucleotide that binds under stringent hybridization conditions to a complement of the nucleotide sequence SEQ ID NO:3, wherein the polynucleotide is at least 30 (e.g., 50, 100, or 200) nucleotides in length.
In a third variation of the composition, the SnSAG-1 marker is an isolated protein including a polypeptide that shares at least 50%, 70%, 90%, or 95% sequence identity with a fragment of the amino acid sequence of SEQ ID NO:1 that is at least 20, 50, 100, or 300 contiguous residues in length. For example, the polypeptide can include at least 20, 50, 100, or 300 contiguous amino acid residues of the sequence of SEQ ID NO:1. The polypeptide can also include the entire amino acid sequence of SEQ ID NO:1. In this composition, the protein can be a fusion protein or a recombinant protein.
In a fourth variation of the composition, the SnSAG-1 marker is a purified antibody that specifically binds to a polypeptide consisting of the amino acid sequence of SEQ ID NO:1, wherein the antibody is a monoclonal antibody or a monospecific polyconal antibody. The purified antibody can be labeled with a detectable label such as a radioisotope, a fluorescent compound, a bioluminescent compound, a chemiluminescent compound, biotin, colloidal gold, a magnetic particle, or an enzyme.
In another aspect, the invention features a method for detecting Sarcocystis neurona in a biological sample. This method includes the steps of: (a) providing the biological sample; and (b) analyzing the biological sample for the presence of a SnSAG-1 marker that is a nucleic acid including a nucleotide sequence that encodes a protein that shares at least 50% sequence identity with SEQ ID NO:1; a polynucleotide that binds under stringent hybridization conditions to a complement of the nucleotide sequence SEQ ID NO:3, wherein the polynucleotide can be at least 30 nucleotides in length; a protein including a polypeptide that shares at least 50% sequence identity with a fragment of the amino acid sequence of SEQ ID NO:1 that can be at least 20 contiguous residues in length; or an antibody that specifically binds to a polypeptide consisting of the amino acid sequence of SEQ ID NO:1. In this method, the presence of the SnSAG-1 marker in the biological sample indicates that the biological sample contains Sarcocystis neurona. 
In the variation of this method where the SnSAG-1 marker is a nucleic acid including a nucleotide sequence that encodes a protein that shares at least 50% sequence identity with SEQ ID NO:1, the nucleotide sequence can be SEQ ID NO:3.
In the variation of this method where the SnSAG-1 marker is a protein including a polypeptide that shares at least 50% sequence identity with a fragment of the amino acid sequence of SEQ ID NO:1 at least 20 contiguous residues in length, the polypeptide can include at least 20 contiguous amino acid residues of the sequence of SEQ ID NO:1. For example, the polypeptide can include the amino acid sequence of SEQ ID NO:1.
The biological sample of the method can include CNS tissue, CSF, blood, or serum. It can also be derived from a horse.
In this method of the inevtnion, the step (B) of analyzing the biological sample for the presence of a SnSAG-1 marker can include isolating RNA from the sample, generating cDNAs from the isolated RNA, and amplifying the cDNAs by PCR to generate a PCR product. The step (B) of analyzing the biological sample for the presence of a SnSAG-1 marker can also include contacting the sample with a labeled oligonucleotide probe that hybridizes under stringent hybridization conditions to the nucleotide sequence SEQ ID NO:3 or a complement of the nucleotide sequence SEQ ID NO:3; or contacting the sample to a molecule that specifically binds to an antibody that specifically binds a protein consisting of the amino acid sequence of SEQ ID NO:1. In the latter, the molecule can be immobilized on a substrate.
In one variation of the method, the biological sample includes SnSAG-1 specific antibodies that are specifically bound to the molecule immobilized on substrate. The antibodies can be detected using a secondary antibody that is labeled with a detectable label. For example, the secondary antibody can specifically bind horse immunoglobulin. The detectable label can be a radioisotope, a fluorescent compound, a bioluminescent compound, a chemiluminescent compound, biotin, colloidal gold, a magnetic particle, or an enzyme (e.g., peroxidase or alkaline phosphatase).
In another variation of this method, the step (B) of analyzing the biological sample for the presence of a SnSAG-1 marker includes contacting the sample with a molecule that specifically binds a protein consisting of the amino acid sequence of SEQ ID NO:1. The molecule can be an antibody such as a monoclonal antibody or a monospecific polyclonal antibody. In this variation, the antibody can be labeled with a detectable label.
The invention also features a composition for stimulating an immune response against Sarcocystis neurona when administered to an animal. The composition includes (a) an isolated agent that can specifically stimulate an immune response against a protein consisting of the amino acid sequence of SEQ ID NO:1 when administered to an animal; and (b) a pharmaceutically acceptable carrier. The composition can include an adjuvant such as an aluminum salt; an oil-in-water emulsion; a composition including saponin; a composition including a bacterial protein; or a cytokine.
The agent that can stimulate an immune response against Sarcocystis neurona when administered to an animal can include a nucleic acid that can be a first polynucleotide including a nucleotide sequence that encodes a protein that shares at least 50% sequence identity with SEQ ID NO:1; or a second polynucleotide that binds under stringent hybridization conditions to a complement of the nucleotide sequence SEQ ID NO:3, wherein the second polynucleotide can be at least 30 nucleotides in length. In the foregoing, the nucleic acid can be a naked DNA, a nucleic acid incorporated into an expression vector, or a polypeptide
Unless otherwise defined, all technical terms used herein have the same meaning as commonly understood by one of ordinary skill in the art to which this invention belongs. Commonly understood definitions of molecular biology terms can be found in Rieger et al., Glossary of Genetics: Classical and Molecular, 5th edition, Springer-Verlag: New York, 1991; and Lewin, Genes V, Oxford University Press: New York, 1994.
By the term xe2x80x9cgenexe2x80x9d is meant a nucleic acid molecule that codes for a particular protein, or in certain cases, a functional or structural RNA molecule. For example, the SnSAG-1 gene encodes the SnSAG-1 protein.
As used herein, a xe2x80x9cnucleic acidxe2x80x9d or a xe2x80x9cnucleic acid moleculexe2x80x9d means a chain of two or more nucleotides such as RNA (ribonucleic acid) and DNA (deoxyribonucleic acid). A xe2x80x9cpurifiedxe2x80x9d nucleic acid molecule is one that is substantially separated from other nucleic acid sequences in a cell or organism in which the nucleic acid naturally occurs (e.g., 30, 40, 50, 60, 70, 80, 90, 95, 96, 97, 98, 99, 100% free of contaminants). The term includes, e.g., a recombinant nucleic acid molecule incorporated into a vector, a plasmid, a virus, or a genome of a prokaryote or eukaryote. Examples of purified nucleic acids include cDNAs, fragments of genomic nucleic acids, nucleic acids produced polymerase chain reaction (PCR), nucleic acids formed by restriction enzyme treatment of genomic nucleic acids, recombinant nucleic acids, and chemically synthesized nucleic acid molecules. A xe2x80x9crecombinantxe2x80x9d nucleic acid molecule is one made by an artificial combination of two otherwise separated segments of sequence, e.g., by chemical synthesis or by the manipulation of isolated segments of nucleic acids by genetic engineering techniques.
By the terms xe2x80x9cSnSAG-1 gene,xe2x80x9d xe2x80x9cSnSAG-1 polynucleotide,xe2x80x9d or xe2x80x9cSnSAG-1 nucleic acidxe2x80x9d is meant a native SnSAG-1-encoding nucleic acid sequence, e.g., the native SnSAG-1 nucleic acid (SEQ ID NO:3); a nucleic acid having sequences from which a SnSAG-1 cDNA can be transcribed; and/or allelic variants and homologs of the foregoing. The terms encompass double-stranded DNA, single-stranded DNA, and RNA.
As used herein, the terms xe2x80x9cproteinxe2x80x9d and xe2x80x9cpolypeptidexe2x80x9d are used synonymously to mean any peptide-linked chain of amino acids, regardless of length or post-translational modification, e.g., glycosylation or phosphorylation. An xe2x80x9cpurifiedxe2x80x9d polypeptide is one that has been substantially separated or isolated away from other polypeptides in a cell, organism, or mixture in which the polypeptide occurs (e.g., 30, 40, 50, 60, 70, 80, 90, 95, 96, 97, 98, 99, 100% free of contaminants). An xe2x80x9cisolatedxe2x80x9d polypeptide is a purified polypeptide that is not included on a substrate (e.g., a polyacrylamide gel) with other purified polypeptides from the cell or organism in which the polypeptide occurs.
By the terms xe2x80x9cSnSAG-1 proteinxe2x80x9d or xe2x80x9cSnSAG-1 polypeptidexe2x80x9d are meant an expression product of a SnSAG-1 nucleic acid (e.g., one consisting of SEQ ID NO:3), or a protein that shares at least 50% (but preferably 55, 60, 65, 70, 75, 80, 85, 90, 95, 96, 97, 98, or 99%) amino acid sequence identity with SEQ ID NO:3.
As used herein, xe2x80x9csequence identityxe2x80x9d means the percentage of identical subunits at corresponding positions in two sequences when the two sequences are aligned to maximize subunit matching, i.e., taking into account gaps and insertions. When a subunit position in both of the two sequences is occupied by the same monomeric subunit, e.g., if a given position is occupied by an alanine in each of two polypeptide molecules, then the molecules are identical at that position. For example, if 7 positions in a sequence 10 amino acids in length are identical to the corresponding positions in a second 10 amino acid sequence, then the two sequences have 70% sequence identity. Likewise, if 7 positions in a sequence 10 nucleotides in length are identical to the corresponding positions in a second 10-nucleotide sequence, then the two sequences have 70% sequence identity. Preferably, the length of the compared sequences is at least 60 nucleotides, more preferably at least 75 nucleotides, and most preferably 100 nucleotides. Sequence identity can be measured using sequence analysis software (e.g., one or more of the algorithms of MegAlign(trademark) sequence analysis software from DNA STAR, Inc., Madison, Wis.).
When referring to hybridization of one nucleic to another, xe2x80x9clow stringency conditionsxe2x80x9d means in 10% formamide, 5xc3x97Denhart""s solution, 6xc3x97SSPE, 0.2% SDS at 42xc2x0 C., followed by washing in 1xc3x97SSPE, 0.2% SDS, at 50xc2x0 C.; xe2x80x9cmoderate stringency conditionsxe2x80x9d means in 50% formamide, 5xc3x97Denhart""s solution, 5xc3x97SSPE, 0.2% SDS at 42xc2x0 C., followed by washing in 0.2xc3x97 SSPE, 0.2% SDS, at 65xc2x0 C.; and xe2x80x9chigh stringency conditionsxe2x80x9d means in 50% formamide, 5xc3x97 Denhart""s solution, 5xc3x97SSPE, 0.2% SDS at 42xc2x0 C., followed by washing in 0.1xc3x97SSPE, and 0.1% SDS at 65xc2x0 C. The phrase xe2x80x9cstringent hybridization conditionsxe2x80x9d means low, moderate, or high stringency conditions.
A xe2x80x9cfragmentxe2x80x9d of a SnSAG-1 nucleic acid is a portion of a SnSAG-1 nucleic acid that is less than full-length and comprises at least a minimum length capable of hybridizing specifically with a native SnSAG-1 nucleic acid under stringent hybridization conditions. The length of such a fragment is preferably at least 15 nucleotides, more preferably at least 20 nucleotides, and most preferably at least 30 nucleotides of a native SnSAG-1 nucleic acid sequence. A xe2x80x9cfragmentxe2x80x9d of a SnSAG-1 polypeptide is a portion of a SnSAG-1 polypeptide that is less than full-length (e.g., a polypeptide consisting of 5, 10, 15, 20, 30, 40, 50, 75, 100 or more amino acids of a native SnSAG-1 protein).
When referring to a nucleic acid molecule or polypeptide, the term xe2x80x9cnativexe2x80x9d refers to a naturally-occurring (e.g., a xe2x80x9cwild-typexe2x80x9d) nucleic acid or polypeptide. A xe2x80x9chomologxe2x80x9d of a SnSAG-1 gene is a gene sequence encoding a SnSAG-1 polypeptide isolated from an organism other than a human being. Similarly, a xe2x80x9chomologxe2x80x9d of a native SnSAG-1 polypeptide is an expression product of a SnSAG-1 gene homolog.
As used herein, a xe2x80x9cSnSAG-1 markerxe2x80x9d is any molecule whose presence in a sample (e.g., a cell) indicates that a SnSAG-1 gene or protein is present in the sample or subject from which the sample was derived. SnSAG-1 markers include SnSAG-1 nucleic acids, SnSAG-1 proteins, and antibodies that specifically bind SnSAG-1 proteins. xe2x80x9cExpressing a SnSAG-1 genexe2x80x9d or like phrases mean that a sample contains a transcription product (e.g., messenger RNA, i.e., xe2x80x9cmRNAxe2x80x9d) of a SnSAG-1 gene or a translation product of a SnSAG-1 protein-encoding nucleic acid (e.g., a SnSAG-1 protein). A cell expresses a SnSAG-1 gene when it contains a detectable level of a SnSAG-1 nucleic acid or a SnSAG-1 protein.
By the term xe2x80x9cantibodyxe2x80x9d is meant an immunoglobulin as well as any portion or fragment of an immunoglobulin whether made by enzymatic digestion of intact immunoglobulin or by techniques in molecular biology. The term also refers to a mixture containing an immunoglobulin (or portion or fragment thereof) such as an antiserum.
The term xe2x80x9cspecifically bindsxe2x80x9d, as used herein, when referring to a polypeptide (including antibodies) or receptor, refers to a binding reaction which is determinative of the presence of the protein or polypeptide or receptor in a heterogeneous population of proteins and other biologics. Thus, under designated conditions (e.g. immunoassay conditions in the case of an antibody), the specified ligand or antibody binds to its particular xe2x80x9ctargetxe2x80x9d (e.g. anti-SnSAG-1 antibody specifically binds to an SnSAG-1polypeptide) and does not bind in a significant amount to other proteins present in the sample or to other proteins to which the ligand or antibody may come in contact in an organism.
By the phrase xe2x80x9cstimulating an immune responsexe2x80x9d is meant eliciting or increasing the activation of a lymphocyte (e.g., a B cell or T cell) or other immune system component. The stimulation of an immune response against a specific antigen can be measured as an increase in antibody titer against that antigen or the activation of one or more lymphocytes having a surface receptor specific for the antigen. Activation of lymphocytes can be determined by conventional assays, e.g., the induction of mitosis, secretion of cytokines, modulation of cell surface molecule expression, secretion of immunoglobulin (B cells), and increased killing of target cells (cytotoxic T cells).
Although methods and materials similar or equivalent to those described herein can be used in the practice or testing of the present invention, suitable methods and materials are described below. All publications, patent applications, patents, and other references mentioned herein are incorporated by reference in their entirety. In the case of conflict, the present specification, including definitions will control. In addition, the particular embodiments discussed below are illustrative only and not intended to be limiting.